Measurement of protein synthesis rates in humans and experimental systems by use of isotopically labeled water

ABSTRACT

Provided herein are methods for measuring protein biosynthesis by using  2 H 2 O or radioactive  3 H 2 O and applicable uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.10/279,399, filed Oct. 23, 2002 now U.S. Pat. No. 7,001,587, titled“MEASUREMENT OF PROTEIN SYNTHESIS RATES IN HUMANS AND EXPERIMENTALSYSTEMS BY USE OF ISOTOPICALLY LABELED WATER”, which claims benefit ofprovisional application Ser. No. 60/335,029, filed on Oct. 24, 2001,both of which are hereby incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was funded in part by grant number AI44767 and AI41401from the National Institutes of Health. The U.S. Government may havecertain rights to this invention.

FIELD OF THE INVENTION

The field of this invention is biochemical kinetics. More specifically,it relates to the measurement of protein synthesis rates.

BACKGROUND OF THE INVENTION

Publications referred to by reference numbering in this specificationcorrespond to the reference list at the end of the specification arehereby incorporated by reference in their entirety.

Control over protein synthesis rates is involved in the regulation ofmost biological processes and is believed to be the primary cause ofnumerous diseases. Regulation of the synthesis rates of biomolecules inliving systems is one of the most fundamental features of biochemicaland physiologic control. For this reason, measurement of biosyntheticrates in vivo has been the subject of enormous research effort over thepast 50 years. Among the macromolecules that have been studied, proteinshave received perhaps the most intense attention due to their centralrole in controlling biological processes. The measurement of proteinsynthesis, as for all other biomolecules, has traditionally required theuse of isotopic labels (stable isotopes or radioisotopes). Many studieshave described isotopic studies of protein biosynthesis (see Waterlow,1978, and Hellerstein & Neese, 1999).

In essence, four general approaches have been described for measuringprotein biosynthetic rates (Waterlow, 1979). These are: (1) exogenouslabeling of proteins of interest, with subsequent re-introduction intothe biological system followed by measurement of die-away curves of thelabeled protein; (2) endogenous pulse-labeling of proteins from alabeled biosynthetic precursor, followed by measurement of die-awaycurves of the labeled proteins of interest; (3) endogenouspulse-labeling of proteins from a labeled biosynthetic precursor,followed by measurement of label incorporation curves into the proteinsof interest, and comparison to estimates of the changing content oflabel present over time in the biosynthetic precursor pool; (4)endogenous labeling of proteins by continuous administration of alabeled biosynthetic precursor, with measurement of label incorporationinto the proteins of interest, and comparison to steady-state labelcontent in the biosynthetic precursor pool (use of precursor-productrelationship).

Among these general labeling strategies, perhaps the most reliabletechnically and operationally is the continuous administration of alabeled biosynthetic precursor (approach #4). This approach takesadvantage of a mathematical principle known as the precursor-productrelationship or, in physics, Newton's cooling equation.

The conceptual basis of the precursor-product relationship is shown inFIG. 1. The central principle is that the label content of the productapproaches a known value, or asymptote, which in turn is determined byand measurable as the label content in the biosynthetic precursor pool.

As summarized by Waterlow et al (1979), this use of theprecursor-product relationship presents several key practical advantagescompared to alternative strategies, particularly when the half-lives ofthe product pool molecules (e.g., proteins) are longer than thehalf-lives of the precursor pool molecules (e.g., free amino acids). Thefirst advantage is that if the isotopic enrichment of the amino acidbiosynthetic precursor pool can be determined and is relatively stableduring the continuous label administration period, only a single timepoint of the protein end-product is, in principle, required tocharacterize the synthesis rate of the protein molecule. This is sobecause the basic precursor-product equation can be used in itsintegrated form when the precursor pool enrichment (S_(A)) is heldsteady:dS _(B) /dt=k(S _(A) −S _(B)).If S _(A) is constant, S _(B(t)) =S _(A)(1−e ^(−kt))or, S _(B(t)) =k[∫₀ ^(t) S _(A) dt−∫ ₀ ^(t) S _(B)(dt)]This relationship is depicted graphically in FIG. 1.

Accordingly, multiple sampling of the protein is not required (unlikedecay curves after endogenous or exogenous labeling) and multiplesampling of the precursor pool is not required (unlike pulse-labelingapproaches). By maintaining a constant or near-constant isotopeenrichment in the precursor pool, problems related to non-steady statecorrections, non-homogeneity or incomplete mixing in the amino acidprecursor pool are also avoided.

Waterlow et al (1979) showed mathematically that synthesis rates arerigorously calculable by this approach even when the protein mass isincreasing or decreasing (i.e., if there is a non-steady state in theend-product pool). This feature allows for broad application of thisapproach, regardless of the physiologic conditions present in the systembeing studied.

There are some practical disadvantages of the continuous administrationapproach, however. The most important of these are: (1) the need forcontinuous administration of the isotopically labeled biosyntheticprecursor, in order to maintain relative steady isotopic enrichments inthe precursor amino acid pool. This requirement typically necessitatescontinuous intravenous infusion or frequent repeated oral dosing overmany days, or even longer. The need for intravenous administrationseverely constrains routine medical diagnostic or field use of thisapproach; (2) the potentially high cost of maintaining a constant levelof label in the biosynthetic precursor pool for a relatively long periodof time; (3) the need to measure the interim isotopic enrichment of thebiosynthetic precursor pool and establish its constancy, and (4)problems in identifying the “true precursor” pool for proteinbiosynthesis in living cells and individuals.

The problem of identifying the true precursor pool for biosynthesisapplies to all applications of the precursor-product relationship, notjust for protein synthesis, and derives from the central principle ofthe technique: the assumption that the labeling curve in the productapproaches a known asymptote, or plateau value, which is determined bythe label content of the precursor pool (FIG. 1). It is thereforeessential to establish during any labeling study the actual asymptoticor plateau value that is being approached. This asymptote value caneither be established by waiting long enough to allow the complete shapeof the labeling curve in the product molecule to become apparent(FIG. 1) or by using a surrogate measure based upon the knownbiochemical organization of the protein biosynthetic system (i.e., fromthe label content in the free amino acid pool leading to proteinsynthesis). However, the biochemical organization of protein synthesisis extremely complex and unpredictable, making the latter approachsubject to significant systematic errors (Waterlow 1979; Airhart 1974;Khairallah and Mortimore 1976).

Alternatively, allowing the shape of the curve to become apparentrequires continuous administration for several half-lives of the proteinend-product. This requirement is most often not practical, in thatprotein half-lives may be several days, weeks or months. It is notpractical to maintain an intravenous infusion for more than 24 to 48hours (even intravenous infusions of this length require medicalpersonnel and monitoring) and oral administration of precursormetabolites cannot achieve stable values in metabolic pools.

Accordingly, it has long been recognized in the field (Waterlow 1979;Hellerstein and Neese 1992; Hellerstein and Neese 1999) that an idealmethod would allow constant isotope levels in the precursor pool to bemaintained for prolonged periods of time in a simple, non-demandingmanner, for example, on the order of a few half-lives of long-livedproteins. However, there has not been a technique that has fulfilledthis objective. A method for measuring protein synthesis that is widelyapplicable, reliable, easy to perform, inexpensive, without toxicitiesor complications, applicable in human subjects, free of the need formedical supervision or in-patient procedures (such as intravenousinfusions), does not require complex instructions, and possesses theadvantages of simple interpretation, therefore would be extremelyvaluable and useful in fields ranging from medical diagnostics to drugdiscovery, genetics, functional genomics, and basic research.

BRIEF SUMMARY OF THE INVENTION

In order to meet these needs, the present invention is directed tomethods of determining the biosynthetic or degradation rate of one ormore proteins or peptides, methods of using the biosynthetic rate and/ordegradation rate determination methods in diagnosis and testing, andkits for determining protein or peptide biosynthetic rates and/ordegradation rates.

In one variation, the invention includes a method of determining thebiosynthetic rate of one or more proteins or peptides in an individualby: (a) administering ²H, ³H, and ¹⁸O labeled water to an individualover a period of time sufficient for the label of the labeled water tobe incorporated into one or more proteins or peptides to form labeledand unlabeled proteins or peptides; (b) obtaining one or more bodilytissues or fluids from the individual, where bodily tissues or fluidsinclude the one or more labeled and unlabeled proteins or peptides; and(c) detecting the incorporation of the label in the one or more labeledproteins or peptides to determine the biosynthetic rate of the one ormore proteins or peptides.

In another variation, the invention includes a continuous labelingmethod of determining the biosynthetic rate of one or more proteins orpeptides in an individual by: (a) administering water labeled with ²H,³H, or ¹⁸O to an individual over a period of time sufficient to maintainrelatively constant water enrichments; (b) obtaining one or more bodilytissues or fluids from the individual, wherein the bodily tissues orfluids comprise the one or more proteins or peptides; (c) measuringincorporation of the label into the one or more proteins or peptides;(d) calculating the isotopic enrichment values of the one or moreproteins or peptides; and (e) applying a precursor-product relationshipto the isotopic enrichment values in order to determine the biosyntheticrate of the one or more proteins or peptides.

In another variation, the isotope enrichment values of the proteins orpeptides may be compared to either to water enrichment values in theindividual or the isotopic plateau value approached by labeled aminoacids.

In another variation, the invention includes a method of determining thebiosynthetic rate of one or more proteins or peptides in an individualby (a) administering ²H, ³H, and/or ¹⁸O labeled water to an individualover a period of time sufficient for the label to be incorporated intothe one or more proteins or peptides and thereby form labeled andunlabeled proteins or peptides; (b) obtaining one or more bodily tissuesor fluids from the individual; (c) hydrolyzing one or more labeled andunlabeled proteins in the one or more bodily tissues or fluids toproduce one or more labeled and unlabeled amino acids; and (d) detectingthe incorporation of the label in the one or more labeled amino acids todetermine the biosynthetic rate of the one or more proteins or peptides.

The present invention is further directed to a method of determining thedegradation rate of one or more proteins or peptides in an individualcomprising the steps of (a) administering ²H, ³H, and/or ¹⁸O labeledwater to an individual over a period of time sufficient for the label tobe incorporated into one or more proteins or peptides to form labeledand unlabeled proteins or peptides; (b) discontinuing the administeringstep (a); (c) obtaining one or more bodily tissues or fluids from theindividual, wherein the bodily tissues or fluids include one or morelabeled and unlabeled proteins or peptides; and (d) detecting theincorporation of the label in the one or more labeled amino acids todetermine the degradation rate of the one or more proteins or peptides.

In another variation, the invention involves a discontinuous labelingmethod for determining the degradation rate of one or more proteins orpeptides in an individual by: (a) administering water labeled with ²H,³H, and ¹⁸O to an individual; (b) discontinuing administering thelabeled water; (c) obtaining one or more bodily tissues or fluids fromthe individual, wherein the bodily tissues or fluids include one or moreproteins or peptides; (d) measuring incorporation of the label into theproteins or peptides; (e) calculating the isotopic enrichment values ofthe one or more proteins or peptides; and (f) applying an exponentialdecay relationship to the isotopic enrichment values to determine thedegradation rate of the one or more proteins or peptides.

In another variation of both continuous and discontinuous labelingmethods, the label is ²H.

In another variation, both continuous and discontinuous labeling methodsmay optionally include partially purifying one or more proteins orpeptides from the bodily tissues or fluids before the measuring step.The partial purification may further include isolating one of the one ormore proteins or peptides.

In another variation, the methods may comprise detecting the one or moreproteins or peptides by mass spectrometry or liquid scintillationcounting. The methods may also optionally be accomplished by massspectrometry alone. In a further variation, the methods may beaccomplished by liquid scintillation counting alone.

In a further variation, the labeled water of both methods may optionallybe administered orally.

In another variation, the measured proteins or peptides include, but arenot limited to, full length proteins or peptide fragments of bonecollagen, liver collagen, lung collagen, cardiac collagen, musclemyosin, serum hormone, plasma apolipoproteins, serum albumin, clottingfactor, immunoglobulin, and mitochondrial protein.

In a further variation, both methods may further include hydrolyzing theone or more proteins or peptides to produce amino acids, and/oroptionally, oligoproteins, prior to measuring isotope incorporation. Theamino acids or oligopeptides may optionally be separated by gaschromatography or HPLC. The gas chromatograph or HPLC may or may not becoupled to the mass spectrometer.

In another variation, the individual of any of the methods is human.

In additional variations, the methods of measuring biosynthetic rates ordegradation rates may be used to diagnose, prognose, or monitordiseases, disorders, and treatment regimens. In one variation, the riskof osteoporosis may be identified by determining the biosynthetic ordegradation rate of bone collagen. In another variation, a response tohormone replacement therapy may be identified by determining thebiosynthetic or degradation rate of bone collagen. In another variation,a response to treatment with a hypolipidemic agent may be identified bydetermining the biosynthetic or degradation rate of apolipoprotein B. Ina further variation, a response to an exercise training or medicalrehabilitation regimen may be identified by determining the biosyntheticor degradation rate of one or more muscle proteins. In yet a furthervariation, an index of hypertrophy versus hyperplasia by measuring theratio of protein:DNA synthesis rates in a tissue may be determined bydetermining the biosynthetic rate or degradation rate. In a furthervariation, the presence or titer of a specific immunoglobulin in anindividual after vaccination or after an infectious exposure may beidentified by determining the biosynthetic rate degradation rate of oneor more immunoglobulins.

In yet another variation, kits for determining the biosynthetic rate ordegradation rate of one or more proteins or peptides in an individualare provided. The kit may include labeled water and instructions for useof the kit. The kit may optionally include chemical compounds forisolating proteins from urine, bone, or muscle, as well as one or moretools for administering labeled water. The kits may further include aninstrument or instruments for collecting a sample from the subject.Procedures employing commercially available assay kits and reagents willtypically be used according to manufacturer defined protocols unlessotherwise noted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the rise-to-plateau principle shown schematically. In FIG.1A, label (*) enters pool A (precursor pool) and pool B (product) issynthesized from A. The replacement rate constant (k) for pool B isrevealed by the shape of the rise-to-plateau curve, as shown here fork=0.1, 0.5 and 1.0 d⁻¹. The plateau value of labeling reached in pool Bwill depend upon the fraction of B derived from the precursor pool.Examples of 50% (FIG. 1B) and 100% B (FIG. 1C) deriving from endogenoussynthesis are shown.

FIGS. 2A-B depict pathways of labeled hydrogen exchange from labeledwater into selected free amino acids. Two NEAA's (alanine, glycine) andan EAA (leucine) are shown, by way of example. Alanine and glycine arepresented in FIG. 2A. Leucine is presented in FIG. 2B. Abbreviations:TA, transaminase; PEP-CK, phosphoenol-pyruvate carbokinase; TCAC,tricarboxylic acid cycle; STHM, serine tetrahydrofolate methyltransferase. FIG. 2C depicts H₂ ¹⁸O labeling of free amino acids forprotein synthesis.

FIG. 3 depicts a schematic model for measurement of new proteinsynthesis from the incorporation of hydrogen-labeled H₂O (*H) intoprotein-bound amino acids. Labeled hydrogens are represented by closedcircles; unlabeled by open circles.

FIG. 4A depicts a schematic model for measurement of new proteinsynthesis from the incorporation of hydrogen-labeled H₂O (*H) intoprotein-bound amino acids. Labeled hydrogens are represented by closedcircles; unlabeled by open circles. The expected time course of labelingeach compartment (body water, free amino acids, protein-bound aminoacids) is shown in the inset (FIG. 4B).

FIG. 5 depicts a flow chart of a method for measuring protein synthesisfrom incorporation of hydrogen-labeled (*H) or oxygen-labeled (*O)H₂O.

FIG. 6 depicts a flow chart of a method for measuring protein synthesisfrom the rate of decline (dilution) of labeled hydrogen in protein-boundamino acids, following washout of hydrogen (*H) or oxygen-labeled(*O)H₂O.

FIG. 7 depicts measured incorporation of ²H₂O into alanine and leucineisolated from rat muscle and heart proteins.

FIG. 8 depicts measured incorporation of ²H₂O into alanine and leucineisolated from rat bone collagen.

FIG. 9 depicts enrichments of ²H₂O in body water of human subjects whodrank 50-100 ml of ²H₂O daily for 10-12 weeks. The data show that theprecursor pool of body water is stable over a period of weeks for eachsubject.

FIGS. 9A and 9C present data collected from healthy subjects. FIGS. 9Band 9D present data collected from HIV/AIDS patients.

FIG. 10 depicts a time course of body ²H₂O enrichments in ratsmaintained on 4% drinking water after baseline priming bolus to 2.5-3.0%body water enrichment.

FIG. 11A depicts a washout of ²H₂O from body ²H₂O in mice afterdiscontinuing ²H₂O administration in drinking water. FIG. 11B depicts awashout of ²H₂O from body ²H₂O in rats after discontinuing ²H₂Oadministration in drinking water.

FIG. 12A depicts die-away curves of ²H-label in rat muscle protein-boundalanine (after discontinuing ²H₂O administration). FIG. 12B depictsdie-away curves of ²H-label in rat muscle protein-bound glutamine (afterdiscontinuing ²H₂O administration). FIG. 12C depicts die-away curves of²H-label in rat muscle protein-bound leucine/isoleucine (afterdiscontinuing ²H₂O administration). FIG. 12D depicts die-away curves of²H-label in rat muscle protein-bound proline (after discontinuing ²H₂Oadministration).

FIG. 13A depicts label incorporation curves into alanine isolated frombone collagen in adult female rats. FIG. 13B depicts label incorporationcurves into glycine isolated from bone collagen in adult female rats.Calculated rate constants for bone collagen synthesis are shown.

FIG. 14A depicts label incorporation curves into alanine isolated fromskeletal muscle proteins in adult female rats. FIG. 14B depicts labelincorporation curves into glutamine isolated from skeletal muscleproteins in adult female rats. FIG. 14C depicts label incorporationcurves into alanine isolated from heart muscle proteins in adult femalerats. Calculated rate constants for protein synthesis are shown.

FIG. 15A depicts label decay curves for alanine isolated from skeletalmuscle of rats, after discontinuing ²H₂O intake in drinking water. FIG.15B depicts label decay curves for alanine isolated from heart muscle ofrats, after discontinuing ²H₂O intake in drinking water. Time zero istwo weeks after discontinuing intake of ²H₂O, as shown in FIG. 11.

DETAILED DESCRIPTION OF THE INVENTION

A method for measuring protein synthesis and degradation rates based onintake of labeled water (²H₂O, ³H₂O, or H₂ ¹⁸O) is described herein.Numerous applications in the fields of medical diagnostics andbiological analysis are discussed.

I. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,immunology, protein kinetics, and mass spectroscopy, which are withinthe skill of the art. Such techniques are explained fully in theliterature, such as, Molecular Cloning: A Laboratory Manual, secondedition (Sambrook et al., 1989) Cold Spring Harbor Press;Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in MolecularBiology, Humana Press; Cell Biology: A Laboratory Notebook (J. E.Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney,ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: LaboratoryProcedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8)J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.);Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell,eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P.Calos, eds., 1987); Current Protocols in Molecular Biology (F. M.Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mulliset al., eds., 1994); Current Protocols in Immunology (J. E. Coligan etal., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons,1999); and Mass isotopomer distribution analysis at eight years:theoretical, analytic and experimental considerations by Hellerstein andNeese (Am J Physiol 276 (Endocrinol Metab. 39) E1146-E1162, 1999).Furthermore, procedures employing commercially available assay kits andreagents will typically be used according to manufacturer-definedprotocols unless otherwise noted.

II. Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The general techniques and procedures described or referencedherein are generally well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, Mass isotopomer distribution analysis at eight years:theoretical, analytic and experimental considerations by Hellerstein andNeese (Am J Physiol 276 (Endocrinol Metab. 39) E1146-E1162, 1999). Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

“Isotopes” refer to atoms with the same number of protons and hence ofthe same element but with different numbers of neutrons (e.g., Hydrogen(H) vs. Deuterium (D)).

“Isotopomers” refer to isotopic isomers or species that have identicalelemental compositions but are constitutionally and/or stereochemicallyisomeric because of isotopic substitution, as for CH₃NH₂, CH₃NHD andCH₂DNH₂.

“Isotopologues” refer to isotopic homologues or molecular species thathave identical elemental and chemical compositions but differ inisotopic content (e.g., CH₃NH₂ vs. CH₃NHD in the example above).Isotopologues are defined by their isotopic composition, therefore eachisotopologue has a unique exact mass but may not have a uniquestructure. An isotopologue is usually comprised of a family of isotopicisomers (isotopomers) which differ by the location of the isotopes onthe molecule (e.g., CH₃NHD and CH₂DNH₂ are the same isotopologue but aredifferent isotopomers).

“Mass isotopomer” refers to a family of isotopic isomers that aregrouped on the basis of nominal mass rather than isotopic composition. Amass isotopomer may comprise molecules of different isotopiccompositions, unlike an isotopologue (e.g., CH₃NHD, ¹³CH₃NH₂, CH₃ ¹⁵NH₂are part of the same mass isotopomer but are different isotopologues).In operational terms, a mass isotopomer is a family of isotopologuesthat are not resolved by a mass spectrometer. For quadrupole massspectrometers, this typically means that mass isotopomers are familiesof isotopologues that share a nominal mass. Thus, the isotopologuesCH₃NH₂ and CH₃NHD differ in nominal mass and are distinguished as beingdifferent mass isotopomers, but the isotopologues CH₃NHD, CH₂DNH₂,¹³CH₃NH₂, and CH₃ ¹⁵NH₂ are all of the same nominal mass and hence arethe same mass isotopomers. Each mass isotopomer is therefore typicallycomposed of more than one isotopologue and has more than one exact mass.The distinction between isotopologues and mass isotopomers is useful inpractice because all individual isotopologues are not resolved usingquadrupole mass spectrometers and may not be resolved even using massspectrometers that produce higher mass resolution, so that calculationsfrom mass spectrometric data must be performed on the abundances of massisotopomers rather than isotopologues. The mass isotopomer lowest inmass is represented as M₀; for most organic molecules, this is thespecies containing all ¹²C, ¹H, ¹⁶O, ¹⁴N, etc. Other mass isotopomersare distinguished by their mass differences from M₀ (M₁, M₂, etc.). Fora given mass isotopomer, the location or position of isotopes within themolecule is not specified and may vary (i.e., “positional isotopomers”are not distinguished).

“Mass isotopomer pattern” refers to a histogram of the abundances of themass isotopomers of a molecule. Traditionally, the pattern is presentedas percent relative abundances where all of the abundances arenormalized to that of the most abundant mass isotopomer; the mostabundant isotopomer is said to be 100%. The preferred form forapplications involving probability analysis, however, is proportion orfractional abundance, where the fraction that each species contributesto the total abundance is used (see below). The term isotope pattern issometimes used in place of mass isotopomer pattern, although technicallythe former term applies only to the abundance pattern of isotopes in anelement.

“Body water enrichment” refers to the percentage of total body waterthat has been labeled upon administration of labeled water.

A “monomer” refers to a chemical unit that combines during the synthesisof a polymer and which is present two or more times in the polymer.

A “polymer” refers to a molecule synthesized from and containing two ormore repeats of a monomer.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, humans, farm animals,sport animals, pets, primates, mice and rats.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual. The definition encompasses blood and other liquidsamples of biological origin, that are accessible from an individualthrough sampling by minimally invasive or non-invasive approaches (e.g.,urine collection, blood drawing, needle aspiration, and other proceduresinvolving minimal risk, discomfort or effort). The definition alsoincludes samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components, such as proteins or polynucleotides.The term “biological sample” also encompasses a clinical sample such asserum, plasma, other biological fluid, or tissue samples, and alsoincludes cells in culture, cell supernatants and cell lysates.

“Biological fluid” includes but is not limited to urine, blood,interstitial fluid, edema fluid, saliva, lacrimal fluid, inflammatoryexudates, synovial fluid, abcess, empyema or other infected fluid,cerebrospinal fluid, sweat, pulmonary secretions (sputum), seminalfluid, feces, bile, intestinal secretions, or other bodily fluid.

“Labeled Water” includes water labeled with a specific heavy isotope ofeither hydrogen or oxygen. Specific examples of labeled water include²H₂O, ³H₂O, and H₂ ¹⁸O.

“Partially purifying” refers to methods of removing one or morecomponents of a mixture of other similar compounds. For example,“partially purifying one or more proteins or peptides” refers toremoving one or more proteins or peptides from a mixture of one or moreproteins or peptides.

“Isolating” refers to separating one compound from a mixture ofcompounds. For example, “isolating one or more proteins or peptides”refers to separating one specific protein or peptide from a mixture ofone or more proteins or peptides.

III. Methods of the Invention

The invention provides general methods for measuring the synthesis rateof proteins in living systems, including long-lived proteins. Thetechnique is based on the exchange of labeled hydrogen or labeled oxygenatoms from water (²H₂O, ³H₂O, or H₂ ¹⁸O) into stable, covalent bonds offree amino acids that are subsequently incorporated into proteins.

As a consequence of these unique features of labeled water and proteinsynthesis, the many technical advantages of a long-term continuous labeladministration (precursor-product) approach for the measurement ofprotein synthesis or protein degradation can be exploited withoutintravenous infusions, medical supervision, special handling needs,sterility concerns, complex instructions, radiation exposure, or highcost. The method is particularly applicable to slow turnover(long-lived) proteins in the body because of the ease with which stablebody water enrichments can be maintained over weeks or months.

A. Administration of Labeled Water

(i) Theory of ²H or ³H-Labeled Water Incorporation

The theory behind ²H₂O or ³H₂O incorporation is as follows:

(1) H₂O availability is probably never limiting for biosyntheticreactions in a cell (because H₂O represents close to 70% of the contentof cells, or >35 Molar concentration), but hydrogen atoms from H₂Ocontribute stochiometrically to many reactions involved in biosyntheticpathways:e.g.: R—CO—CH₂—COOH+NADPH+H₂O→R—CH₂CH₂COOH (fatty acid synthesis).

As a consequence, label provided in the form of H-labeled water isincorporated into biomolecules as part of synthetic pathways. Hydrogenincorporation can occur in two ways: into labile positions in a molecule(i.e., rapidly exchangeable, not requiring enzyme catalyzed reactions)or into stable positions (i.e., not rapidly exchangeable, requiringenzyme catalysis).

(2) Some of the hydrogen-incorporating steps from cellular water intoC—H bonds in biomolecules only occur during well-definedenzyme-catalyzed steps in the biosynthetic reaction sequence, and arenot labile (exchangeable with solvent water in the tissue) once presentin the mature end-product molecules. For example, the C—H bonds onglucose are not exchangeable in solution. In contrast, each of thefollowing C—H positions exchanges with body water during reversal ofspecific enzymatic reactions: C-1 and C-6, in the oxaloacetate/succinatesequence in the Krebs' cycle and in the lactate/pyruvate reaction; C-2,in the glucose-6-phosphate/fructose-6-phosphate reaction; C-3 and C-4,in the glyceraldehyde-3-phosphate/dihydroxyacetone-phosphate reaction;C-5, in the 3-phosphoglycerate/glyceraldehyde-3-phosphate andglucose-6-phosphate/fructose-6-phosphate reactions (Katz 1976).

Labeled hydrogen atoms from water (²H₂O or ³H₂O) that are covalentlyincorporated into specific non-labile positions of a molecule therebyreveals the molecule's “biosynthetic history”—i.e., label incorporationsignifies that the molecule was synthesized during the period that ²H₂Oor ³H₂O was present in cellular water.

(3) The labile hydrogens (non-covalently associated or present inexchangeable covalent bonds) in these biomolecules do not reveal themolecule's biosynthetic history. Labile hydrogen atoms can be easilyremoved by incubation with unlabelled water (H₂O) (i.e., by reversal ofthe same non-enzymatic exchange reactions through which ²H or ³H wasincorporated in the first place), however:

As a consequence, potentially contaminating ²H or ³H label that does notreflect biosynthetic history but is incorporated via non-syntheticexchange reactions, can easily be removed in practice by incubation withnatural abundance H₂O.

(4) Analytic methods are available for measuring quantitatively theincorporation of labeled hydrogen atoms into biomolecules (e.g., liquidscintillation counting for ³H; mass spectrometry or NMR spectroscopy for²H). For further discussions on the theory of labeled waterincorporation, see, for example, Jungas 1968.

(ii) Incorporation of Isotopes from Labeled Water into Amino Acids

a) Hydrogen Isotopes (²H₂O and ³H₂O)

The hydrogen atoms on C—H bonds are the hydrogen atoms on amino acidsthat are the most useful for measuring protein synthesis from ²H₂O sincethe O—H and N—H bonds of peptides and proteins are labile in aqueoussolution. As such, the exchange of ²H-label from ²H₂O into O—H or N—Hbonds occurs without the synthesis of proteins from free amino acids asdescribed above. C—H bonds undergo exchange from H₂O into free aminoacids during specific enzyme-catalyzed intermediary metabolic reactions(FIG. 2). The presence of ²H-label in C—H bonds of protein-bound aminoacids after ²H₂O administration therefore means that the protein wasassembled from amino acids that were in the free form during the periodof ²H₂O exposure—i.e. that the protein is newly synthesized.Analytically, the amino acid derivative used must contain all the C—Hbonds but must remove all potentially contaminating N—H and O—H bonds.

The key question that needs to be answered is the degree of labelingpresent in C—H bonds of free amino acid or, more specifically, intRNA-AA, during exposure to ²H₂O in body water. The total number of C—Hbonds in each NEAA is known—e.g. 4 in alanine, 2 in glycine, etc. (FIG.2).

Hydrogen atoms from body water may be incorporated into free aminoacids. ²H or ³H from labeled water can enter into free amino acids inthe cell through the reactions of intermediary metabolism, but ²H or ³Hcannot enter into amino acids that are present in peptide bonds or thatare bound to transfer RNA. Free essential amino acids may incorporate asingle hydrogen atom from body water into the α-carbon C—H bond, throughrapidly reversible transamination reactions (FIG. 2). Free non-essentialamino acids contain a larger number of metabolically exchangeable C—Hbonds, of course, and are therefore expected to exhibit higher I.E.(isotopic enrichment) values per molecule from ²H₂O in newly synthesizedproteins (FIGS. 2A-B).

One of skill in the art will recognize that labeled hydrogen atoms frombody water may be incorporated into other amino acids via otherbiochemical pathways. For example, it is known in the art that hydrogenatoms from water may be incorporated into glutamate via synthesis of theprecursor α-ketoglutrate in the citric acid cycle. Glutamate, in turn,is known to be the biochemical precursor for glutamine, proline, andarginine. Other amino acids synthesis pathways are known to those ofskill in the art.

b) Oxygen Isotopes (H₂ ¹⁸O)

Oxygen atoms may also be incorporated into amino acids throughenzyme-catalyzed reactions. For example, oxygen exchange into thecarboxylic acid moiety of amino acids may occur during enzyme catalyzedreactions. Incorporation of labeled oxygen into amino acids is known toone of skill in the art as illustrated in FIG. 2C.

B. Determination of Protein Biosynthesis and Degradation Rates

Protein biosynthesis and degradation rates can be determined by twogeneral methods using the techniques of this invention: continuouslabeled water administration or discontinuous labeled wateradministration. For continuous labeled water administration, thefollowing generalized steps may be followed: (a) administering labeledwater to an individual over a period of time sufficient to maintainrelatively constant water enrichments over time in the individual,wherein the water is labeled with one or more isotopes such as ²H, ³H,and ¹⁸O; (b) obtaining one or more bodily tissues or fluids from theindividual; (c) measuring incorporation of the one or more isotopes intothe one or more proteins or peptides; (d) calculating the isotopicenrichment values in the one or more proteins or peptides; and (e)applying a precursor-product relationship to the isotopic enrichmentvalues to determine the biosynthetic rate of the one or more proteins orpeptides. Optionally, the proteins or peptides may be partiallypurified, or fully isolated, from the biological sample samplecollected. Furthermore, the component amino acids of the proteins orpeptides may be isolated and analyzed.

For the discontinuous labeled water administration, the followinggeneralized steps may be followed: (a) administering labeled water to anindividual, wherein the water is labeled with one or more isotopes suchas ²H, ³H, and ¹⁸O; (b) discontinuing the administering step (a); (c)obtaining one or more bodily tissues or fluids from the individual; (d)measuring incorporation of the one or more isotopes into the one or moreproteins or peptides; (e) calculating the isotopic enrichment values inthe one or more proteins or peptides; and (f) applying an exponentialdecay relationship to the isotopic enrichment values to determine thedegradation rate of the one or more proteins or peptides. Optionally,the proteins or peptides may be partially purified or isolated from thesample. Furthermore, the component amino acids of the proteins orpeptides may be isolated and analyzed.

It should be noted that the above steps need not be conducted in theexact order specified. For example, the isotopic enrichment values maybe calculated prior isolating bodily tissues or measuring isotopicincorporation into proteins or peptides.

(i) Administering Labeled Water to an Individual

For both methods, labeled water (particularly ²H₂O, ³H₂O, or H₂ ¹⁸O) maybe readily obtained commercially. ²H₂O may be purchased from CambridgeIsotope Labs (Andover, Mass.). ³H₂O may be purchased, e.g., from NewEngland Nuclear, Inc. Chemicals and enzymes may be purchased from Sigma,Inc. (St. Louis, Mo.). In general, ²H₂O is non-radioactive and thus,presents less toxicity concerns than radioactive ³H₂O. If ³H₂O isutilized, then a non-toxic amount, which is known to those of skill inthe art, is administered.

Relatively high body water enrichments of ²H₂O (e.g., 1-5% of the totalbody water is labeled) have been achieved using the techniques of theinvention. This water enrichment is relatively constant and stable asthese levels are maintained for weeks or months in humans and inexperimental animals without any evidence of toxicity (see FIGS. 3 and9-10). This finding in a large number of human subjects (>100 people) iscontrary to previous concerns about vestibular toxicities at high dosesof ²H₂O. Applicants have discovered that as long as rapid changes inbody water enrichment are prevented (e.g., by initial administration insmall, divided doses), high body water enrichments of ²H₂O can bemaintained with no toxicities. Previous dosing of ²H₂O in humans, forother purposes (e.g., measurement of fatty acid or cholesterol synthesisor gluconeogenesis) have typically used lower doses and achieved lowerbody water enrichment. The low expense of commercially available ²H₂Oallows long-term maintenance of enrichments in the 1-5% range atrelatively low expense (e.g., lower cost for 2 months labeling at 2%²H₂O enrichment, and thus 7-8% enrichment in the alanine precursor pool(FIGS. 2A-B), than for 12 hours labeling of ²H-leucine at 10% freeleucine enrichment, and thus 7-8% enrichment in leucine precursor poolfor that period).

Relatively high and relatively constant body water enrichments foradministration of H₂ ¹⁸O may also be accomplished, since the ¹⁸O isotopeis not toxic, and does not present a significant health risk as a result(FIG. 2C).

Administration of labeled water can be achieved in various ways. For thecontinuous labeling method, sufficient amount of labeled water isadministered such that an isotopic plateau value of maximal or isotopicenrichment is approached (i.e. wherein the concentration of labeledwater is relatively constant over time). For example, see FIG. 4, *Henrichment v. time. If the continuous labeling period can be maintainedfor as long as 4-5 half-lives of a protein, the asymptote reached andthe shape of the I.E. curve approaching this asymptote will reveal the“true precursor” isotopic enrichment [3] as well as the fractionalreplacement rate of the protein product (FIG. 1). By labeling to plateauwhile maintaining a stable precursor pool enrichment, it is therebypossible to overcome the biological complexities of cellular amino acidpools.

In one embodiment, labeled water such as ²H₂O is taken orally (e.g., bydrinking via the mouth) intermittently to achieve a relatively constantenrichment in the individual. In another embodiment, labeled water isadministered intravenously to achieve a relatively constant waterenrichment in the individual. This method of administration avoidsfrequent oral dosing. In another embodiment, the duration of labeledwater exposure is sufficient to characterize the full isotopeincorporation curve into the protein approaching its asymptotic value orsufficient to characterize the full isotope incorporation curve into adifferent, fully or nearly fully turned-over protein. Once dailyadministration of small amounts of ²H₂O (3-6 ounces/day) allowsmaintenance of extremely constant levels of ²H₂O enrichment in bodywater in humans (FIG. 3) and administration in drinking water allowsconstant levels in animals (FIG. 4) for periods as long as severalmonths or longer. This stability is due to the uniquely slow turnover ofthe body water pool compared to any other biosynthetic precursor inanimals (e.g., half-life of 20 minutes for ²H-glucose, <30 minutes for²H-leucine, versus 10-14 days for ²H₂O).

For the discontinuous labeling method, an amount of labeled water ismeasured and then administered, one or more times, and then the exposureto labeled water is discontinued and wash-out of labeled water from bodywater pool is allowed to occur. The time course of protein degradationmay then be monitored.

(ii) Obtaining One or More Bodily Tissues or Fluids from Said Individual

For both continuous labeling and discontinuous labeling methods, abiological sample is obtained from bodily fluids or tissues of anindividual. Specific methods of obtaining biological samples are wellknown in the art. Bodily fluids include, but are not limited to, urine,blood, blood serum, amniotic fluid, spinal fluid, conjunctival fluid,salivary fluid, vaginal fluid, stool, seminal fluid, and sweat. Thefluids may be isolated by standard medical procedures known in the art.

One or more proteins or peptides may be obtained, and optionallypartially purified or isolated, from the biological sample usingstandard biochemical methods known in the art. Examples of proteins thatmay be partially purified or isolated include, but are not limited to,bone collagen, liver collagen, lung collagen, cardiac collagen, musclemyosin, serum hormone, plasma apolipoproteins, serum albumin, clottingfactor, immunoglobulin, mitochondrial protein. Peptide fragments ofproteins may also be obtained. The frequency of biological sampling canvary depending on different factors. Such factors include, but are notlimited to, the nature of the one or more proteins or peptides, ease ofsampling, half-life of a drug used in a treatment if monitoringresponses to treatment.

For both continuous and discontinuous labeling methods, the one or moreproteins and/or peptides may also be purified partially purified, oroptionally, isolated, by conventional purification methods includinghigh pressure liquid chromatography (HPLC), fast performance liquidchromatography (FPLC), gas chromatography, gel electrophoresis, and/orany other separation methods.

For both continuous and discontinuous labeling methods, the one or moreproteins and/or peptides may be hydrolyzed to form smaller oligopeptidesor amino acids. Hydrolysis methods include any method known in the art,including, but not limited to, chemical hydrolysis (such as acidhydrolysis) and biochemical hydrolysis (such as peptidase degradation).Hydrolysis may be conducted either before or after protein and/orpeptide purification and/or isolation. The oligopeptides and amino acidsalso may be partially purified, or optionally, isolated, by conventionalpurification methods including high performance liquid chromatography(HPLC), fast performance liquid chromatography (FPLC), gaschromatography, gel electrophoresis, and/or any other methods ofseparating chemical compounds, proteins, peptides, or amino acids.

(iii) Detecting the Incorporation of One or More Isotopes

For both continuous and discontinuous labeling methods, isotopicenrichment from proteins, peptides or amino acids can be determined byvarious methods such as mass spectrometry, particularity gaschromatography-mass spectrometry (GC-MS), nuclear magnetic resonance(NMR), or liquid scintillation counting.

Incorporation of labeled isotopes into proteins or peptides may bemeasured directly. Alternatively, incorporation of labeled isotopes maybe determined by measuring the incorporation of labeled isotopes intoone or more oligopeptide or amino acid hydrolysis products of peptidesand proteins. The hydrolysis products may optionally be measuredfollowing either partial purification or isolation by any knownseparation method, as described previously.

a. Mass Spectrometry

Mass spectrometers convert components of a sample into rapidly movinggaseous ions and separate them on the basis of their mass-to-chargeratios. The distributions of isotopes or isotopologues of ions, or ionfragments, may thus be used to measure the isotopic enrichment in one ormore protein, peptide, or amino acid.

Generally, mass spectrometers comprise an ionization means and a massanalyzer. A number of different types of mass analyzers are known in theart. These include, but are not limited to, magnetic sector analyzers,electrostatic analyzers, quadrapoles, ion traps, time of flight massanalyzers, and fourier transform analyzers. In addition, two or moremass analyzers may be coupled (MS/MS) first to separate precursor ions,then to separate and measure gas phase fragment ions.

Mass spectrometers may also include a number of different ionizationmethods. These include, but are not limited to, gas phase ionizationsources such as electron impact, chemical ionization, and fieldionization, as well as desorption sources, such as field desorption,fast atom bombardment, matrix assisted laser desorption/ionization, andsurface enhanced laser desorption/ionization. In addition, massspectrometers may be coupled to separation means such as gaschromatography (GC) and high performance liquid chromatography (HPLC).In gas-chromatography mass-spectrometry (GC/MS), capillary columns froma gas chromatograph are coupled directly to the mass spectrometer,optionally using a jet separator. In such an application, the gaschromatography (GC) column separates sample components from the samplegas mixture and the separated components are ionized and chemicallyanalyzed in the mass spectrometer.

When GC/MS is used to measure mass isotopomer abundances of organicmolecules such as amino acids, hydrogen-labeled isotope incorporation isamplified 3 to 7-fold. This is because of the nearly linear additiveeffects of hydrogen-labeling in each labeling location of an amino acid(FIG. 2). As a consequence of this discovery, the sensitivity andefficiency of hydrogen labeling attains to levels comparable toadministration of specifically labeled amino acids.

In one embodiment, ²H, ³H, or ¹⁸O-enrichments of proteins or peptidesmay be measured directly by mass spectrometry.

In another embodiment, the proteins or peptides may be partiallypurified, or optionally isolated, prior to mass spectral analysis.Furthermore, the component amino acids of the polypeptides may bepurified.

In another embodiment, ²H, ³H, or ¹⁸O-enrichments of proteins, peptides,amino acids after hydrolysis of a protein or peptide, are measured bygas chromatography-mass spectrometry.

In each of the above embodiments, because of the unique constancy ofbody labeled water enrichments over time, the synthesis rate of theprotein can be calculated by application of the precursor-productrelationship using either labeled body water enrichment values orasymptotic isotope enrichment in the relevant amino acid of a fullyturned over protein to represent the true precursor pool enrichment.Alternatively, the degradation rate may be calculated using anexponential decay curve.

b. Liquid Scintillation

Radioactive isotopes may be observed using a liquid scintillationcounter. Radioactive isotopes such as ³H emit radiation that is detectedby a liquid scintillation detector. The detector converts the radiationinto an electrical signal, which is amplified. Accordingly, the numberof radioactive isotopes in a protein, peptide, or amino acid may bemeasured.

In one embodiment, the ³H-enrichment value in a bodily tissue or fluidmay be measured directly by liquid scintillation.

In another embodiment, the proteins or peptides or component amino acidsmay be partially purified, or optionally isolated, and subsequentlymeasured by liquid scintillation.

In another embodiment ³H-enrichments of proteins, peptides, amino acidsafter hydrolysis of the protein or peptide, are measured by liquidscintillation. In each of the above embodiments, because of the uniqueconstancy of body labeled water enrichments over time, the synthesisrate of the protein can be calculated by application of theprecursor-product relationship using either labeled body waterenrichment values or asymptotic isotope enrichment in the relevant aminoacid of a fully turned over protein to represent the true precursor poolenrichment. Alternatively, the degradation rate may be calculated usingan exponential decay curve.

(iv) Determining the Biosynthetic or Degradation Rate

a. MIDA: Where p Reflects Body H₂O Enrichment—Calculating theRelationship between Mass Isotopomer Abundances and p in a Polymer ofKnown n

Biosynthetic and degradation rates may be calculated by combinatorialanalysis, by hand or via an algorithm. Variations of Mass IsotopomerDistribution Analysis (MIDA) combinatorial algorithm are discussed in anumber of different sources known to one skilled in the art.Specifically, the MIDA calculation methods are the subject of U.S. Pat.No. 5,336,686, incorporated herein by reference. The method is furtherdiscussed by Hellerstein and Neese (1999), as well as Chinkes, et al.(1996), and Kelleher and Masterson (1992), all of which are herebyincorporated by reference in their entirety.

In addition to the above-cited references, calculation softwareimplementing the method is publicly available from Professor MarcHellerstein, University of California, Berkeley.

In brief, calculation of the number (n) of metabolically exchangedH-atoms (between amino acids and cellular water) is by combinatorialanalysis, or MIDA. In brief, the relative fraction of double-labeled tosingle-labeled amino acid molecules reveals n if the precursor poolenrichment of ²H, ³H, or ¹⁸O (p) is known. If one assumes that preflects body labeled water enrichment, then n can be calculated bycombinatorial analysis.

Fractional abundances of mass isotopomers result from mixing naturalabundance molecules with molecules newly synthesized from a pool oflabeled monomers characterized by the parameter p. A mixture of thistype can be fully characterized by f, the fraction new, and p. Thealgorithm proceeds in step-wise fashion, beginning with the simplestcalculation, a molecule synthesized from a single element containingisotopes with the same fractional abundances that occur in nature andnot mixed with any other molecules. We then proceed to moleculescontaining more than one element with all isotopes at natural abundance;then to non-polymeric molecules containing different elements, some ofwhich are in groups whose isotope composition is not restricted tonatural abundance but is variable; then to polymeric moleculescontaining combinations of repeating chemical units (monomers), whereinthe monomers are either unlabeled (containing a natural abundancedistribution of isotopes) or potentially labeled (containing anisotopically-perturbed element group); and finally to mixtures ofpolymeric molecules, composed of both natural abundance polymers andpotentially labeled polymers, the latter containing combinations ofnatural abundance and isotopically-perturbed units.

The last-named calculation addresses the condition generally present ina biological system, wherein polymers newly synthesized during theperiod of an isotope incorporation experiment are present along withpre-existing, natural abundance polymers and the investigator isinterested in determining the proportion of each that is present, inorder to infer synthesis rates or related parameters.

The calculation may be accomplished for full-length proteins or peptidesprior to hydrolysis. Alternatively, the calculation may be conducted foramino acid hydrolysis products (e.g. oligopeptides or amino acids) ofthe proteins or peptides following hydrolysis.

iv. b. Where Partial Exchange of Labeled H-Atoms is Assumed—CalculatingExpressing the Relationship Between Mass Isotopomer Abundances and n ata Known p

Alternatively, all the potentially exchanging H-atoms in each amino acid(e.g. 2 for glycine, 4 for alanine, 5 for glutamine, etc.) could beassumed to be partially exchanged with water and the isotopic enrichmentof the total H-atom pool (p) calculated by MIDA.

The same MIDA algorithms disclosed in (a) above to generate tablesexpressing the relationship between mass isotopomer abundances and p ina polymer of known n can be used for expressing the relationship betweenmass isotopomer abundances and n at a known p. Both types of tables areshown for glycine and alanine (Table 1A-C).

TABLE 1A Example of MIDA tables generated for selected NEAA (alanine andglycine), including two different values of n for alanine. Alanine, n =3 p M₀ M₁ M₂ EM₁ EM₂ Ratio 0.000 0.8944 0.0955 0.0101 0.002 0.88910.1003 0.0106 0.0048 0.0005 0.1090 0.004 0.8838 0.1050 0.0111 0.00950.0011 0.1112 0.006 0.8786 0.1097 0.0117 0.0142 0.0016 0.1135 0.0080.8733 0.1144 0.0123 0.0189 0.0022 0.1157 0.010 0.8681 0.1190 0.01290.0235 0.0028 0.1180 0.012 0.8629 0.1236 0.0135 0.0281 0.0034 0.12030.014 0.8578 0.1281 0.0141 0.0326 0.0040 0.1226 0.016 0.8526 0.13260.0147 0.0371 0.0046 0.1249 0.018 0.8475 0.1371 0.0154 0.0416 0.00530.1273 0.020 0.8424 0.1415 0.0161 0.0460 0.0060 0.1296 0.022 0.83730.1459 0.0167 0.0504 0.0067 0.1319 0.024 0.8323 0.1503 0.0174 0.05480.0074 0.1343 0.026 0.8273 0.1546 0.0182 0.0591 0.0081 0.1367 0.0280.8222 0.1589 0.0189 0.0634 0.0088 0.1390 0.030 0.8173 0.1631 0.01970.0676 0.0096 0.1414 0.032 0.8123 0.1673 0.0204 0.0718 0.0103 0.14380.034 0.8073 0.1715 0.0212 0.0760 0.0111 0.1462 0.036 0.8024 0.17560.0220 0.0801 0.0119 0.1487 0.038 0.7975 0.1797 0.0228 0.0842 0.01270.1511 0.040 0.7926 0.1837 0.0236 0.0882 0.0135 0.1535 0.042 0.78780.1877 0.0245 0.0922 0.0144 0.1560 0.044 0.7829 0.1917 0.0253 0.09620.0152 0.1585 0.046 0.7781 0.1957 0.0262 0.1002 0.0161 0.1609 0.0480.7733 0.1996 0.0271 0.1041 0.0170 0.1634 0.050 0.7686 0.2034 0.02800.1079 0.0179 0.1659

TABLE 1B Example of MIDA tables generated for selected NEAA (alanine andglycine), including two different values of n for alanine. Alanine, n =4 p M₀ M₁ M₂ EM₁ EM₂ Ratio 0.000 0.8944 0.0955 0.0101 0.002 0.88740.1019 0.0108 0.0064 0.0007 0.1101 0.004 0.8803 0.1081 0.0115 0.01260.0014 0.1135 0.006 0.8734 0.1143 0.0123 0.0188 0.0022 0.1169 0.0080.8664 0.1205 0.0131 0.0250 0.0030 0.1203 0.010 0.8596 0.1265 0.01390.0310 0.0038 0.1237 0.012 0.8527 0.1325 0.0148 0.0370 0.0047 0.12710.014 0.8459 0.1384 0.0157 0.0429 0.0056 0.1306 0.016 0.8392 0.14420.0166 0.0487 0.0065 0.1341 0.018 0.8325 0.1499 0.0176 0.0544 0.00750.1376 0.020 0.8258 0.1556 0.0186 0.0601 0.0085 0.1411 0.022 0.81920.1612 0.0196 0.0657 0.0095 0.1446 0.024 0.8127 0.1667 0.0206 0.07120.0105 0.1481 0.026 0.8061 0.1722 0.0217 0.0767 0.0116 0.1517 0.0280.7996 0.1775 0.0228 0.0820 0.0127 0.1553 0.030 0.7932 0.1828 0.02400.0873 0.0139 0.1589 0.032 0.7868 0.1881 0.0251 0.0926 0.0150 0.16250.034 0.7805 0.1932 0.0263 0.0977 0.0162 0.1661 0.036 0.7741 0.19830.0275 0.1028 0.0175 0.1698 0.038 0.7679 0.2033 0.0288 0.1078 0.01870.1734 0.040 0.7617 0.2083 0.0301 0.1128 0.0200 0.1771 0.042 0.75550.2132 0.0314 0.1177 0.0213 0.1808 0.044 0.7493 0.2180 0.0327 0.12250.0226 0.1845 0.046 0.7432 0.2227 0.0340 0.1272 0.0240 0.1882 0.0480.7372 0.2274 0.0354 0.1319 0.0253 0.1920 0.050 0.7312 0.2320 0.03680.1365 0.0267 0.1958

TABLE 1C Example of MIDA tables generated for selected NEAA (alanine andglycine), including two different values of n for alanine. Glycine, n =2 p M₀ M₁ M₂ EM₁ EM₂ Ratio 0.000 0.9045 0.0863 0.0092 0.002 0.90090.0896 0.0095 0.0033 0.0003 0.0952 0.004 0.8973 0.0928 0.0098 0.00650.0006 0.0963 0.006 0.8937 0.0961 0.0102 0.0098 0.0010 0.0974 0.0080.8902 0.0993 0.0105 0.0130 0.0013 0.0985 0.010 0.8866 0.1025 0.01080.0162 0.0016 0.0996 0.012 0.8831 0.1057 0.0112 0.0194 0.0020 0.10080.014 0.8796 0.1089 0.0115 0.0226 0.0023 0.1019 0.016 0.8760 0.11210.0119 0.0258 0.0027 0.1030 0.018 0.8725 0.1153 0.0122 0.0289 0.00300.1042 0.020 0.8690 0.1184 0.0126 0.0321 0.0034 0.1053 0.022 0.86550.1215 0.0130 0.0352 0.0038 0.1065 0.024 0.8620 0.1247 0.0133 0.03830.0041 0.1077 0.026 0.8585 0.1278 0.0137 0.0415 0.0045 0.1088 0.0280.8550 0.1309 0.0141 0.0445 0.0049 0.1100 0.030 0.8515 0.1339 0.01450.0476 0.0053 0.1112 0.032 0.8481 0.1370 0.0149 0.0507 0.0057 0.11230.034 0.8446 0.1401 0.0153 0.0538 0.0061 0.1135 0.036 0.8412 0.14310.0157 0.0568 0.0065 0.1147 0.038 0.8377 0.1461 0.0162 0.0598 0.00690.1159 0.040 0.8343 0.1492 0.0166 0.0628 0.0074 0.1171 0.042 0.83080.1522 0.0170 0.0658 0.0078 0.1183 0.044 0.8274 0.1551 0.0174 0.06880.0082 0.1195 0.046 0.8240 0.1581 0.0179 0.0718 0.0087 0.1207 0.0480.8206 0.1611 0.0183 0.0748 0.0091 0.1220 0.050 0.8172 0.1640 0.01880.0777 0.0096 0.1232 Values shown represent fractional abundances,normalized for the sum of the M₀ to M₂ - mass isotopomers measured.Abbreviations: n, number of exchanging hydrogen atoms in C—H bonds ofNEAA (see text). EM₁ and EM₂ excess fractional abundance of M₁ and M₂mass isotopomers, after subtraction of natural abundance (p = 0.000)value. The values of EM₁ and EM₂ shown here representmaximal values(i.e., f = 100%), or A₁ ^(∞) and A₂ ^(∞) (14); Ratio, ratio of EM₂/EM₁.Calculation algorithms are described in detail elsewhere (14). Foralanine, n = 3 and n = 4 sample tables are shown. A table fornon-integral values of n can also be generated for glycine.Calculations are for the N-acetyl, n-butyl ester derivative of eachamino acid. In practice, these MIDA “training tables” can be used toconvert measured mass isotopomer ratios (e.g. excess M₂:excessM₁[EM₂/EM₁]) into the n or p present; then, using this value of n or p,the asymptotic label achievable in the most abundant mass isotopomer(e.g. A₁ ^(∞)) is determined for calculation of fractional synthesis.Sample calculations for the different models (as described above) areshown (Table 1).

Some examples of experimental data are shown for glycine and alaninefrom bone collagen in an animal (Table 2).

TABLE 2 Representative labeling data in bone collagen from a rat given4% ²H₂O in drinking water for 3 weeks Body ²H₂O EM₂/EM₁ Enrichment EM₁EM₂ Ratio (%) Calculated n A₁ ^(∞) f(%) Protein-bound glycine 0.02040.0022 0.1078 2.6% 2 0.0390 52.3 Protein-bound alanine 0.0395 0.00600.1518 2.6% 4 0.0767 51.5 Experimental results and calculations areshown for bone collagen isolated from a rat after 3 weeks of ²H₂O intake(4%) in drinking water. EM₁ and EM₂ were measured in alanine and glycinefrom the collagen hyrolysate, as described in the text. The value of nfor alanine and glycine were calculated based on measured body ²H₂Oenrichment (2.6%), using table 1. (see text for details). Thecalculatedvalue of n and measured ²H₂O enrichment were then used tocalculate A₁ ^(∞)(asymptotic EM₁ value, if 100% new synthesis, ref 14).Comparison of measured EM₁ to calculated A₁ ^(∞) allows calculation offractional synthesis (f).Alanine EM₁=0.0395 and EM₂=0.0060 after 3 wk of ²H₂O labeling. Body ²H₂Oenrichments were stable at 2.6%. The EM₂/EM₁ ratio is 0.1518, whichaccording to table 1 is consistent with n=4 at ²H₂O=2.6% (model 1).Because the maximal n for alanine (4) is estimated to represent 100% ofthe measured body ²H₂O enrichment (2.6%) used for p, there is no need tocompare different models of incomplete exchange of alanine hydrogen withbody ²H₂O (i.e. exchange is complete). The calculated fractionalsynthesis of bone collagen is 51.5%. Results for glycine from the sameanimal revealed an EM₂/EM₁ ratio of 0.1078 (EM₂=0.002, EM₁=0.024),consistent with n=4 at ²H₂O=2.42% (Table 1), and fractional synthesis of52.8%.

iv. c. Comparison of Methods (a) and (b)

The model disclosed in (a) above generates an estimate of activelyexchanging H-atoms (e.g. ¾ for alanine), similar to the approach at Leeet al. The model disclosed in (b) above generates a fraction of each C—Hposition that has been exchanged with body water (e.g. 75% exchange inall H's in alanine). It turns out that these two models generate verysimilar kinetic calculations. Based on the calculated n of C—H positionsexchanging with body water in each amino acid (AA), a maximal orasymptotic label incorporation into each mass isotopomer (A_(x) ^(∞)) isthen calculated, using the standard MIDA formulae. This asymptotic valuerepresents the denominator for calculating fractional synthesis fromlabel incorporation curves. Confirmation of these MIDA-calculated A_(x)^(∞) values can then be achieved by long-term labeling protocols.

iv. (d) Applying the Precursor Product Relationship

Next, for the continuous labeling method, the isotopic enrichment iscompared to asymptotic (i.e., maximal possible) enrichment and theprotein or peptide kinetic parameters (e.g., protein biosynthesis rates)are calculated from precursor-product equations. For the discontinuouslabeling method, the rate of decline in isotope enrichment is calculatedand the protein or peptide kinetic parameters are calculated fromexponential decay equations.

The fractional synthesis or replacement rate (k_(s)) of proteins orpeptides may be determined by application of the continuous labeling,precursor-product formula:

${{k_{s}\left( d^{- 1} \right)} = {{- \left\lbrack {\ln\left( {1 - \frac{{{AA}({protein})}_{t}\left( {I.E.} \right)}{A^{\infty}\left( {I.E.} \right)}} \right)} \right\rbrack}/{{time}(d)}}},$where A^(∞) represents the asymptotic or plateau value of the amino acidI.E. (isotopic enrichment) possible under the labeling conditionspresent and AA(protein)_(t) represents the measured protein-bound aminoacid at time t.

Alternatively, the fractional synthesis or replacement rate (k_(s)) ofproteins or peptides may be determined by comparing isotope enrichmentvalues of one or more proteins or peptides to either water enrichmentvalues in the individual. For degradation studies, fractionaldegradation rates (k_(d)) were calculated by the standard exponentialdecay equation:

${{k_{d}\left( d^{- 1} \right)} = {{- \left\lbrack {\ln\left( \frac{{{AA}({protein})}_{t}\left( {I.E.} \right)}{{A({protein})}_{0}\left( {I.E.} \right)} \right)} \right\rbrack}/{{time}(d)}}},$where AA(protein)₀ represents the measured protein-bound AA at time zeroand AA(protein)_(t) represents the measured protein-bound AA at time t.

Advantages of Labeling of Proteins from Labeled Water

Use of a long-term constant labeled water enrichment approach in factprovides several enormous practical and technical advantages for themeasurement of protein synthesis, that had not previously beenrecognized. These advantages arise because this method allows use of theprecursor-product method in its classic, asymptotic form (FIG. 1):S _(B(t)) =S _(A)(1−e ^(−kt))

(1) The option of sampling only one (or a small number) of time pointsin the product pool is provided, because constant precursor poolenrichments are reliably maintained (FIGS. 3 and 4).

(2) The constant precursor pool enrichments of the “constant infusion”approach are achieved without the need for intravenous infusions orfrequent oral dosing regimens, and without the need for medicalsupervision, complex instructions, special refrigeration or handlingneeds, sterility testing, etc. Indeed, there can be few if any long-termlabeling protocols for human subjects as easy and convenient as drinkinga few ounces of water every day or every several days. The ease of thisapproach represents an enormous operational advantage.

(3) Because of the extreme ease of labeled water administration, thelack of need for medical supervision or facilities, and the low cost of²H₂O labeling, very long-term labeling protocols (i.e., not just hoursor even days, but weeks or months) are permitted. Comparable long-termlabeling protocols using specific labeled amino acid precursors are notfeasible.

(4) The option of convenient very long-term labeling protocols usingthis version of the “continuous administration” precursor-product methodpermits studies of very-slow-turnover proteins, which include some ofthe most important and interesting proteins in the body, such as bonecollagen (the key factor in osteoporosis), muscle myosin (the key factorin strength and rehabilitation therapy), immunoglobulins (the basis ofhumoral immunity), etc. These studies would not be feasible oraffordable in humans using a rapidly turning-over labeled precursormolecule, such as a labeled amino acid.

(5) The option of long-term labeling protocols using the “continuousadministration” precursor-product method permits full characterizationof the product labeling curve up to the plateau or asymptotic labelingvalues, thereby overcoming the central methodologic problem ofalternative label incorporation studies for protein biosynthesis(problems in estimating the true precursor pool enrichment, Waterlow1979). Documenting the actual asymptotic value attained is the mostrigorously correct solution, in principle, to the problem of the trueprecursor pool, and this solution becomes available with the labeledwater administration method (FIGS. 2, 3 and 4).

(6) Costs are several orders of magnitude lower than equivalentlong-term labeling with specific amino acids (e.g., 1 L of ²H₂O givenover 6 weeks at a cost of about $170 is equivalent to several kg of ²Hor ¹³C labeled alanine or glycine to achieve the same isotopicenrichments for 6 weeks, at a cost of many thousands of dollars.

(7) Incorporation of labeled isotopes (e.g. ²H, ³H, and ¹⁸O) fromlabeled water into amino acids is highly reproducible. For example,incorporation of ²H from ²H₂O into the C—H bonds of the NEAA studied(alanine or glycine) was highly reproducible (Table 1). Thederivatization procedure, combined with the isolation procedure of AA'sand proteins, which involves incubation in aqueous solution, removeslabile hydrogens present in O—H and N—H bonds. The long period oflabeled water administration allows sufficient time for consistentincorporation into both C-2 and other positions of NEAA's to occur. Massisotopomer analysis confirmed the near-complete exchange of C—Hpositions in NEAA's such as alanine and glycine. Thus, administration oflabeled water in essence results in as reliable labeling as continuousadministration of exogenously labeled AA's.

(8) The capacity to achieve 1-2% body water enrichments in humans aswell as rodents without toxicities or side-effects, combined with theamplification factor introduced by multiple sites of potential hydrogenentry (FIG. 2), and the analytic precision and sensitivity of massspectrometers, makes labeling amino acids via labeled water a veryefficient, rather than an inefficient, approach. Label incorporationcurves can be precisely characterized even at relatively low body waterenrichments (e.g. 0.25-0.50%). It is worth pointing out that theamplification factor derives from multiple C—H bonds being potentiallylabeled and only applies for mass isotopomer analysis of intact AAmolecules, i.e. not for radioactivity measurements or forcombustion/isotope ratio approaches.

(9) The ease of oral labeled water administration obviates the need forintravenous infusions, medical supervision, sterility concerns, specialhandling of tracers, or complex instructions. Field studies are madepractical, even for long-lived proteins.

(10) The unique constancy of body labeled water enrichments over time(FIG. 10), due to the large and slowly turning over body water pool,makes this approach ideal for application of the rise-to-plateau orprecursor-product relationship to slowly turning over proteins. Combinedwith the extreme ease of oral labeled water administration and therelatively low cost of labeled water (e.g. ²H₂O), very slow turnoverproteins can be studied by this technique. Our measurements of bonecollagen synthesis (FIGS. 13 and 14) and mixed muscle protein synthesis(FIG. 15) are examples of this application.

(11) The ease and low cost of continuous labeling with labeled water,especially ²H₂O, permits full exploitation of the rise-to-plateauapproach, because constant water labeling is feasible for >4-5half-lives of almost any protein of interest. This is apparent in thestudies of bone collagen synthesis. Variable dilution within tRNA-AApools [1-5] is overcome by tracing the product labeling curve up to itsplateau value (FIG. 1). This possibility represents an importanttheoretical advantage over most alternative attempts to estimate tRNA-AA(true precursor) enrichments (Waterlow, 1978).

IV. Methods of Use

Using the methods disclosed herein, protein kinetic parameters such asprotein biosynthesis or degradation rates can be determined from anynumber of protein in an individual. These rates can be applied fordiagnostic and/or monitoring uses. Many research and clinicalapplications of this technique can be envisioned, including determiningsynthesis and turnover rates of medically important proteins such asmuscle or cardiac myosin; bone, liver, lung or cardiac collagen; serumhormones; plasma apolipoproteins; serum albumin, clotting factors, andother proteins; immunoglobulins; mitochondrial proteins.

Other uses include, but are not limited to, measurement of bone collagensynthesis as an index of osteoporotic risk, measurement of bone collagensynthesis to monitor responses to hormone replacement therapy, both ofwhich are based on the incorporation of isotopes from labeled water intoamino acids in bone collagen. Synthesis of tissue collagen can be usedas a measure of fibroproliferative rate in disorders such as livercirhosis, interstitial lung disease, congestive heart failure,sclerodoma, coal miner's pneumonia (black lung), kidney fibrosis, andother diseases of fibrogenesis and fibrolysis. Response to anti-fibroticagent therapy can be monitored by the change in tissue collagensynthesis. A patient's progress after treatment with a hypolipidemicagent can be monitored by measuring apolipoprotein B synthesis (e.g., anHMG-CoA reductase inhibitor), based on the incorporation of isotopesfrom labeled water into alanine or other amino acids in apolipoproteinB. An individual's response to an exercise training or medicalrehabilitation regimen can be monitored by measuring the synthesis andbreakdown rates of muscle proteins, based on the incorporation ofisotopes from labeled water into amino acids in muscle proteins. Anindex of hypertrophy versus hyperplasia can be determined by measuringthe ratio of protein: DNA synthesis rates in a tissue. The presenceand/or titer of specific immunoglobulins after vaccination or after aninfectious exposure can be determined by measuring the synthesis rate ofspecific immunoglobulins, based on the incorporation of isotopes fromlabeled water into immunoglobulin subpopulations.

In another aspect, the invention provides kits for analyzing proteinsynthesis rates in vivo. The kits may include labeled water(particularly ²H₂O, ³H₂O, and H₂ ¹⁸O labeled water or a combinationthereof), and in preferred embodiments, chemical compounds known in theart for isolating proteins from urine, bone, or muscle and/or chemicalsnecessary to get a tissue sample, automated calculation software forcombinatorial analysis, and instructions for use of the kit.

Other kit components, such as tools for administration of water (e.g.,measuring cup, needles, syringes, pipettes, IV tubing), may optionallybe provided in the kit. Similarly, instruments for obtaining samplesfrom the subject (e.g., specimen cups, needles, syringes, and tissuesampling devices may also be optionally provided.

V. Literature Citations

-   1. Airhart, J., A. Vidrich, and E. A. Khairallah. Compartmentation    of free amino acids for protein synthesis in rat liver. Biochem J    140: 539-45, 1974.-   2. Bonotto, S., I. Ndoite, G. Nuyts, E. Fagniart, and R. Kirchmon.    Study of the distribution and biological effects of ³H in the Algae    Acetabularia, Chlamydomonas and Porphyra. Curr Top Rad Quart 12:    115-132, 1977.-   3. Etnier, E., C. Travis, and D. Hetrick. Metabolism of organically    bound tritium in man. Rad Res 100: 487-502, 1984.-   4. Hellerstein, M. Methods for measurement of polymerization    biosynthesis: three general solutions to the problem of the “true    precursor”. Diabetes, Nutrition and Metabolism In Press, 2000.-   5. Hellerstein, M. K., and R. A. Neese. Mass isotopomer distribution    analysis at eight years: theoretical, analytic, and experimental    considerations. Am J Physiol 276: E1146-62, 1999.-   6. Hellerstein, M. K., and R. A. Neese. Mass isotopomer distribution    analysis: a technique for measuring biosynthesis and turnover of    polymers. Am J Physiol 263: E988-1001, 1992.-   7. Humphrey, T., and D. Davies. A new method for the measurement of    protein turnover. Biochem J 148: 119-127, 1975.-   8. Humphrey, T., and D. Davies. A sensitive method for measuring    protein turnover based on the measurement of 2-³H-labeled amino    acids in proteins. Biochem J 156: 561-568, 1976.-   9. Jungas, R. L. Fatty acid synthesis in adipose tissue incubated in    tritiated water. Biochemistry 7: 3708-17, 1968.-   10. Katz, J., and R. Rognstad. Futile cycles in the metabolism of    glucose. Curr Top Cell Regul 10: 237-89, 1976.-   11. Khairallah, E. A., and G. E. Mortimore. Assessment of protein    turnover in perfused rat liver. Evidence for amino acid    compartmentation from differential labeling of free and tRNA-gound    valine. J Biol Chem 251: 1375-84, 1976.-   12. Mather-Devré, R., and J. Binet. Molecular aspects of tritiated    water and natural water in radiation biology. Prog Biophys Molec    Biol 43: 161-193, 1984.-   13. Mewissen, D., M. Furedi, A. Ugarte, and J. Rust. Comparative    incorporation of tritium from tritiated water vs tritiated    thymidine, uridine or leucine. Curr Top Rad Res Quart 12: 225-254,    1977.-   14. Waterlow, J. C., P. J. Garlick, and D. J. Millward, eds. 1978.    Protein Turnover in Mammalian Tissues and in the Whole Body. North    Holland, Amsterdam.-   15. Patterson, B. W., and R. R. Wolfe. Concentration dependance of    methyl-palmitate isotope ratios by electron impact ionization gas    chromatography/mass spectrometry. Bioi. Mass Spectrom. 22: 481-486,    1993.-   16. Kelleher, J. K., and T. M. Masterson. Model equations for    condensation biosynthesis using stable isotopes and radioisotopes.    Am. J. Physiol. 262 (Endocrinol. Metab. 25): E118-E125, 1992.-   17. Chinkes, D. L., A. Aarsland, J. Rosenblatt, and R. R. Wolfe.    Comparison of mass isotopomer dilution methods used to calculate    VLDL production in vivo. Am. J. Physiol. 271 (Endocrinol. Metab.    34): E373-E383, 1996.

EXAMPLES Example 1 ²H₂O Labeling in Rat

Sprague-Dawley rats (200-250 g, Simonsen Inc., Gilroy Calif.) andC57Blk/6ksj mice (10-15 g, Jackson Laboratories, Bar Harbor Me.) wereused. Housing was in individual cages for rats and groups of 5 for mice.Feeding was ad-libitum with Purina® rodent chow. All studies receivedprior approval from the UC Berkeley Animal Care and Use Committee.

The ²H₂O labeling protocol consisted of an initial intraperitoneal (ip)injection of 99.9% ²H₂O, to achieve ca. 2.5% body water enrichment(assuming total body water to be 60% of body weight) followed byadministration of 4% ²H₂O in drinking water. For labeling of rats inutero, 4% drinking water was started while the male and female adultrats were housed together for mating (i.e. before pregnancy) and the 4%²H₂O drinking water was continued through the pregnancy andpost-delivery period.

Urine was collected longitudinally from some animals to establish thetime course of body ²H₂O enrichments. A de-labeling protocol was alsocarried out in some animals, to monitor label die-away in body water andin proteins. After completing 8-10 weeks of ²H₂O labeling, the 4% ²H₂Owas replaced with unlabeled drinking water. Rats were then sacrificedweekly, to establish the time course of body water enrichment and muscleprotein labeling. The de-labeling period was for 6 weeks.

Ovariectomy was performed in adult female rodents as described (9).After allowing 3 weeks for recovery from surgery, the rats receivedeither estradiol by subcutaneous pellet (200 μg) or sham placement ofpellet. ²H₂O labeling was initiated at the time of pellet placement andcontinued for 2 weeks, at which time the animals were sacrificed andbone was collected.

Example 2 Incorporation of ²H₂O in Rat Muscle

FIG. 7 depicts measured incorporation of ²H₂O into selected amino acidsisolated from muscle proteins in the rat. Enrichments of ²H in aminoacids was determined by gas chromatographic-mass spectrometric analysisafter hydrolysis of muscle proteins to free amino acids.

Example 3 Incorporation of ²H₂O in Rat Bone Collagen

FIG. 8 depicts measured incorporation of ²H₂O into selected amino acidsisolated from bone collagen in the rat. Enrichment of ²H in amino acidswas determined by gas chromatographic-mass spectrometric analysis afterhydrolysis of bone collagen to free amino acids. Calculated collagenturnover rate constant (k) was nearly identical from glycine (k=0.044d⁻¹), alanine (k=0.041 d⁻¹), or proline (not shown, k=0.038 d⁻¹).

Rats were killed every 1-2 weeks during a 10-week period of ²H₂Oadministration. The rear left femur was collected and was dissected freeof soft tissue. Bone marrow and trabecular bone were removed using aneedle with sharp cutting surface (ref: Bone 2000). After washing 3times with water, the bone was splintered and powdered under liquid N₂in a Spex mill and defatted with chloroform:methanol (1:1, v:v). Afterdrying, the powdered bone was subjected to acid hydrolysis in 6N HCl(110° C., 24 hr). The free AA were dried under N₂ gas and derivatizedfor analysis by gas chromatography/mass spectrometry (GC/MS).

Example 4 Incorporation of ²H₂O in Humans

FIG. 9 depicts enrichments of ²H₂O in body water of human subjects whodrank 50-100 ml of ²H₂O daily for 10-12 weeks. Left, healthy subjects;right, HIV/AIDS patients. No adverse effects or toxicities were observedin any subjects. Body ²H₂O enrichments were measured by a gaschromatographic-mass spectrometric technique. Constant water enrichmentlevels over time were observed for each patient.

Example 5 Incorporation of ²H₂O in Body Water of Rats

FIG. 10 depicts the enrichment of ²H₂O in body water of rats given 4%²H₂O as drinking water. Animals grew normally and exhibited no signs oftoxicity. Body ²H₂O enrichments were measured by GC/MS.

The time course of ²H incorporation from ²H₂O into AA's from bonecollagen was measured in growing, adult mice (FIG. 13). The rateconstant for rise to plateau (k_(s)) was similar for the NEAA tested(e.g. k_(s(ala))=0.178 wk⁻¹, k_(s(glyc))=0.163 wk⁻¹).

Example 6 Discontinuous ²H₂O Administration in Rats

FIG. 11 depicts a washout of ²H₂O from body ²H₂O in rats and mice afterdiscontinuing ²H₂O administration in drinking water. Kinetic informationcan also be inferred from the label decay curves after cessation of ²H₂Oadministration. Turnover of body water pools is relatively slow (FIG.11), so that true label dilution did not begin until 10-14 days afterdiscontinuing ²H₂O administration (FIG. 15). Subsequent die-away curves(e.g. from weeks 3-6) reveal k_(d). The rate constants calculated weresimilar to values obtained from label incorporation studies (e.g.k_(d)=0.19 wk⁻¹ for alanine in skeletal muscle proteins and k_(d)=0.37wk⁻¹ for alanine in heart muscle proteins, FIG. 15).

Example 7 Discontinuous ²H₂O Administration in Rats for Measuring RatMuscle Rates

FIG. 12 depicts die-away curves of ²H-label in rat muscle protein-boundamino acids (after discontinuing ²H₂O administration). Mixed proteinswere isolated from hindlimb muscle (quadriceps femoris) and heart during8-10 weeks of ²H₂O administration (4% in drinking water) followed by a6-week de-labeling period (re-institution of unlabeled drinking water).Rats were killed weekly (n=3/group) during the labeling and delabelingperiods and skeletal muscle and heart were collected. Tissues werefrozen in liquid N₂ at the time of sacrifice. Mixed proteins werehydrolyzed to free amino acids in 6N HCl, as described elsewhere (10).Amino acids were then derivatized for GC/MS analysis.

Example 8 Measurement of ²H₂O Enrichment of Body Water by GC/MS

The ²H₂O enrichment of body water was measured by a GC/MS technique thatwe have described elsewhere (Neese et al., Analytic Biochem 298(2):189-95, 2001). Briefly, the hydrogen atoms from water (10 μL) werechemically transferred to acetylene by reaction with calcium carbide ina sealed vial. Acetylene gas was then derivatized by injection intoanother sealed vial containing 0.5 ml Br₂ (0.1 mM) dissolved in CCl₄,followed by quenching of remaining Br₂ with cyclohexene. The resultingtetrabromoethane was dissolved in CCl₄ and was analyzed by GC/MS, usinga DB-225 column (30 m, J&W, Folsom, Calif.) at 180° C., with methanechemical ionization (C.I.). The ions at m/z 265 and 266 were analyzedusing selected ion monitoring. These ions represent the M₀ and M₁ massisotopomers of the C₂H₂Br₃ ⁺ fragment (⁷⁹Br⁷⁹Br⁸¹Br isotopologue). Theenrichments of ²H₂O in water samples were calculated by comparison tostandard curves generated by mixing 100% ²H₂O with unlabeled H₂O inknown proportions (Neese et al., Analytic Biochem 298(2): 189-95, 2001).

Example 9 Measurement of Isotope Abundances of AA's by GC/MS

The mass isotopomer abundances of AA were analyzed as the N-acetyl,n-butyl ester derivative. Retention times of individual AA wereestablished by use of unlabeled standards. The M₀-M₂ ions were analyzedfor each NEAA, by selected ion monitoring (Table 1). The column was aDB225 at 120-220° C., with methane C.I. Adjustment of injection volumeswas performed to maintain abundances of each NEAA within a range thatallowed accurate measurement of isotope abundances.

Example 10 Number of Exchanging C—H Positions in AA and Determination ofA₁ ^(∞)

Two independent approaches were used for determining A_(x) ^(∞) (themaximal isotopic enrichment of a particular mass isotopomer in aprotein-bound AA during a continuous labeling protocol): combinatorialanalysis (MIDA) and labeling to 100% replacement.

The ratio of excess double-labeled (EM₂):excess single-labeled (EM₁) AAmolecules reflects the isotopic enrichment of exchanging H-atoms (p) andthe number of H-atoms actively exchanging (n), in accordance withprinciples of combinatorial probabilities. The ratio of EM₂/EM₁ invarious non-essential AA was therefore measured as a means ofcalculating n (Table 1 and FIG. 2).

Table 3 shows the results of these experiments.

TABLE 3 Calculated values of n in free amino acids isolated from bonecollagen in rats after ²H₂O intake. Alanine Glutamine Body water EM₂/EM₁Calculated EM₂/EM₁ Calculated ²H₂O Ratio n Ratio n 0.0300 0.1493 3.450.2261 4.75 0.0290 0.1514 3.67 0.2287 5.03 0.0300 0.1514 3.57 0.22754.82 0.0310 0.1534 3.60 0.2352 5.10 0.0270 0.1531 3.97 0.2380 5.900.0310 0.1551 3.69 0.2314 4.90 0.0280 0.1504 3.70 0.2278 5.13 0.03100.1563 3.76 0.2293 4.79 0.0280 0.1498 3.66 0.2261 5.03 0.0280 0.15493.98 0.2372 5.67 0.0270 0.1508 3.83 0.2234 5.02 0.0290 0.1543 3.830.2252 4.83 0.0290 0.1518 3.69 0.2235 4.74 0.0290 0.1532 3.77 0.21844.45 Mean ± S.D. 3.73 ± 0.15 5.01 ± 0.37 Rats were given 4% ²H₂O asdrinking water for 5–11 weeks. Bone collagen was isolated and hydrolyzedto free AA as described in text and EM₂/EM₁ ratios were measured inhydrolysate alanine an glutamine. The measured body water enrichment ineach animal was used to calculate a table of n as a function oftheoretical EM₂/EM₁ ratios, using integral values of n (i.e., n = I, 2,3, 4, 5), for each AA.The measured EM₂/EM₁ ratio in each AA was thenplotted on the best-fit regression line calculated from the integralvalues of n, to calculate the best-fit value of n in the animal.The MIDA-calculated values of actively exchanging H-positions weresimilar for bone collagen, muscle protein and in utero-labeled mixedproteins and revealed near-complete exchange for certain NEAA (e.g.calculated n for alanine≅4, for glycine≅2) under all the experimentalconditions studied.

Isotope enrichments were measured in protein-bound alanine and glycinein different tissues isolated from rat pups labeled with ²H₂O in utero(Table 4).

TABLE 4 Measured enrichments and calculated value of p and n forprotein-bound glycine and alanine from different tissues in rat pupsexposed to ²H₂O in utero. EM₂/EM₁ Animal Tissue EM₁ EM₂ Ratio Calc. P(n) Protein-Bound Glycine 1 Liver 0.0403 0.0044 0.1088 2.60% (2) Muscle0.0396 0.0042 0.1071 2.30% (2) Brain 0.0410 0.0044 0.1071 2.30% (2) 2Liver 0.0403 0.0044 0.1088 2.60% (2) Muscle 0.0388 0.0042 0.1082 2.55%(2) Brain 0.0412 0.0045 0.1104 2.83% (2) 3 Liver 0.0425 0.0046 0.10742.38% (2) Brain 0.0427 0.0047 0.1071 2.25% (2) Mean ± SD 0.0408 ± 0.00130.0044 ± 0.0002 0.1086 2.48 ± 0.20% (2) Protein-Bound Alanine 1 Liver0.0821 0.0124 0.1510 2.57% (4) Muscle 0.0824 0.0126 0.1529 2.67% (4)Brain 0.0711 0.0103 0.1449 2.22% (4) 2 Liver 0.0827 0.0126 0.1524 2.64%(4) Brain 0.0691 0.0099 0.1433 2.33% (4) Mean ± SD 0.0783 ± 0.00680.0116 ± 0.0013 0.1489 ± 0.0045 2.49 ± 0.20 (4) A female rat was startedon 4% ²H₂O in drinking water just prior to mating. The dam wasmaintained on 4% ²H₂O throughout pregnancy and delivery. Within 24 hr ofdelivery, the mother and 3 pups were sacrificed. Blood was collectedfrom the mother for measurement of body ²H₂O enrichment. The pups weredissected and samples of liver, muscle and brain tissue were collected.Mixed proteins from thesetissues were precipitated and hydrolyzed tofree amino acids, as described in the text. Isotope enrichments inprotein-bound glycine and alanine are shown, with calculated values of p(²H-enrichment of exchanging H-atoms in the C—H backbone), assumingmaximal values of n (i.e. full exchange). Alternatively, the mother's²H₂O enrichment was used to calculate n based on the measuredEM₂/EM₁)ratio; the nearest integral value calculated for each sample is shown inparentheses. The mother's body ²H₂O enrichment was 2.4%.The calculated values of p for H-atoms entering each NEAA were veryclose to the measured body ²H₂O enrichment in the mother at the time ofsacrifice (2.49±0.20% calculated from alanine and 2.48±0.20% fromglycine, compared to 2.4% from the measured ²H₂O enrichment in maternalblood), when n=4 was used for the value of exchanging H-atoms in alanineand n=2 was used in glycine (i.e. complete exchange). Based on thesecalculated values of p and n, average protein fractional synthesis inthese in utero-labeled animals was as expected, about 100% (99.8% forprotein-bound glycine, 106% for protein-bound alanine).

These results support the validity of the combinatorial calculations andthe underlying model for calculating p and n.

Example 11 Time Course of ²H-AA Labeling in Bone Collagen and MixedMuscle Proteins

The time course of ²H incorporation from ²H₂O into amino acids from bonecollagen was measured in growing, adult mice (FIG. 13). The rateconstant for rise to plateau (k_(s)) was similar for the NEAA tested(e.g. k_(s(ala))=0.178 wk⁻¹, k_(s(glyc))=0.163 wk⁻¹).

Administration of estrogen pellets (200 μg) to ovariectomized femalerats resulted in a ca. 35-40% decrease in k_(s) of bone collagen,compared to vehicle-implanted, ovariectomized rats (FIG. 14) from 0.012to 0.008 d⁻¹. ²H incorporation into mixed proteins isolated fromskeletal muscle was consistent for different AA. Values of k_(s) forskeletal muscle were 0.21 wk⁻¹ (alanine) and 0.23 wk⁻¹ (glutamine).Replacement rates of mixed proteins from heart were higher (e.g.k_(s)=0.31 wk⁻¹, for alanine, in heart muscle proteins).

Example 12 In Vitro Studies with ²H₂O

After incubation of an unlabeled protein (human serum albumin) in 70%²H₂O for 24 hr at room temperature and subsequent hydrolysis to free AA,no ²H incorporation was observed in any of the derivatized AA. Moreover,when ²H₁-labeled alanine (C—H bond labeled in carbon-2) or ²H₂-labeledglycine (C—H bond labeled in carbon-2) were subjected to theacid-hydrolysis conditions used for proteins, no loss of ²H-label wasobserved.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application. Allpublications, patents and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent or patent applicationwere specifically and individually indicated to be so incorporated byreference.

1. An isolated, ²H labeled peptide or protein molecule with its ²H labelderived from ²H labeled water (²H₂O), wherein said isolated, ²H labeledpeptide or protein molecule is produced by administering the ²H₂O to anindividual wherein said ²H₂O is labeled with a detectable amount of ²H,wherein the ²H is incorporated into the peptide or protein molecule ofthe individual via metabolic synthesis, and isolating said ²H labeledpeptide or protein molecule wherein the labeled peptide or proteinmolecule comprises a full length protein or a peptide fragment selectedfrom the group consisting of bone collagen, liver collagen, lungcollagen, cardiac collagen, muscle myosin, serum hormone, plasmaapoliprotein, serum albumin, clotting factor, immunoglobulin andmitochondrial protein.
 2. The isolated, labeled peptide or proteinmolecule of claim 1, wherein the ²H₂O is administered to the individualover a period of time sumcient to maintain relatively constant labeledbody water enrichments.
 3. The isolated, labeled peptide or proteinmolecule of claim 1, wherein the ²H₂O is administered to the individualfor a period of time, prior to discontinuing the administering of the²H₂O.
 4. The isolated, labeled peptide or protein molecule of claim 1,wherein the ²H₂O is administered orally to the individual.
 5. Theisolated, labeled peptide or protein molecule of claim 1, wherein the²H₂O is administered intravenously to the individual.
 6. The isolated,labeled peptide or proteim molecule of claim 1, wherein the individualto whom the ²H₂O is administered is human.
 7. The isolated, labeledpeptide or protein molecule of claim 1 wherein said labeled peptide orprotein molecule is obtained from bodily tissues or fluids from theindividual.
 8. The isolated, labeled peptide or protein molecule ofclaim 1, wherein the labeled peptide or protein molecule is isolatedusing mass spectrometry, liquid scintillation counting, or a combinationof mass spectrometry and liquid scintillation counting.
 9. The isolated,labeled peptide or protein molecule of claim 1, wherein the labeledpeptide or protein molecule is separated by purification methods chosenfrom high pressure liquid chromatography (HPLC), fast performance liquidchromatography (FPLC), gas chromatography, gel electrophoresis, and anycombination thereof.
 10. The isolated, labeled peptide or proteinmolecule of claim 1, wherein the full length protein or peptide fragmentis bone collagen.
 11. The isolated, labeled peptide or protein moleculeof claim 1, wherein the full length protein or peptide fragment is livercollagen.
 12. The isolated, labeled peptide or protein molecule of claim1, wherein the full length protein or peptide fragment is lung collagen.13. The isolated, labeled peptide or protein molecule of claim 1,wherein the full length protein or peptide fragment is cardiac collagen.14. The isolated, labeled peptide or protein molecule of claim 1,wherein the full length protein or peptide fragment is muscle myosin.15. The isolated, labeled peptide or protein molecule of claim 1,wherein the full length protein or peptide fragment is serum hormone.16. The isolated, labeled peptide or protein molecule of claim 1,wherein the full length protein or peptide fragment is plasmaapoliprotein.
 17. The isolated, labeled peptide or protein molecule ofclaim 1, wherein the full length protein or peptide fragment is serumalbumin.
 18. The isolated, labeled peptide or protein molecule of claim1, wherein the full length protein or peptide fragment is clottingfactor.
 19. The isolated, labeled peptide or protein molecule of claim1, wherein the full length protein or peptide fragment isimmunoglobulin.
 20. The isolated, labeled peptide or protein molecule ofclaim 1, wherein the full length protein or peptide fragment is amitochondrial protein.